Cat+-independent phospholipase A2 (iPLA2) activity is elevated during myocardial ischemia. Investigating the regulation and function of iPLA2 will help to elucidate the physiological and pathological role of this enzyme in myocardium. In order to analyze the biochemical properties of iPLA2, the multiple isoforms of this enzyme will be expressed and purified using a baculovirus expression system. Following enzyme purification, enzyme kinetics and factors which modulate iPLA2 activity will be studied. Multiple iPLA2 mutants will also be constructed and expressed in this system so that the residues critical for enzymatic activity may be identified. The identification of these critical residues may ultimately aid in the design and selection of specific inhibitors, which potentially could be used as pharmacotherapeutics. Another important aim of this study is to investigate the regulation of iPLA2. Additionally, a rat cardiomyocyte-derived cell line (H9c2) will be utilized to study the role of iPLA2 in a model of cardiac ischemia and in cell signaling. Following simulated ischemia or activation with IL-1(3, H9c2 cells will be assayed for increased iPLA2 activity. If altered activity is evident, upstream modulators and downstream effects of increased iPLA2 activity will be investigated. Thus, the aim of these studies is to understand the regulation and function of iPLA2 in cardiomyocyte signaling and ischemia.